Electron microscopic and histochemical studies of the mononuclear odontoclast of the
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THE ANATOMICAL RECORD 240:42-51 (1994)
Electron Microscopic and Histochemical Studies of the Mononuclear
Odontoclast of the Human
TAKANORI DOMON, KAZUYUKI SUGAYA, YASUTAKA YAWAKA,
MASAKAZU OSANAI, YOSHINORI HANAIZUMI, SHIGERU TAKAHASHI,
AND MINORU WAKITA
Departments of Oral Anatomy 11 (T.D., Y.H., S.T., M.W.) and Pediatric Dentistry (Y.Y.,
M.O.), Hokkaido University School of Dentistry, Sapporo, Department of Anatomy 11
(K.S.), Nihon University School of Dentistry at Matsudo, Matsudo, Japan
ABSTRACT
Background: Osteoclasts and odontoclasts are multinucleated giant cells which resorb hard tissue by the ruffled borders. Recently,
the authors reported the presence of a mononuclear osteoclast with a ruffled border in vitro. However, its presence in vivo has not been shown. To
demonstrate the presence of a mononuclear odontoclast in humans, the
present study used human deciduous teeth.
Methods: After fixation and decalcification, tartrate-resistant acid phosphatase (TRACPase) activity was detected with the azo dye method, and
then TRACPase-positive cells were observed on resorbing areas of teeth.
TRACPase-positive cells could be distinguished from other cells by light
microscopy, and the cells for investigation were serially sectioned by alternating semithin and ultrathin sections to observe their ultrastructure and
three-dimensional organization.
Results: TRACPase activity was detected in both multinucleated odontoclasts and a mononuclear cell from serial sections. By electron microscopy,
most of the multinucleated odontoclasts had ruffled borders and clear
zones. A mononuclear TRACPase-positive cell with a ruffled border and
clear zone was reconstructed three-dimensionally by NIKON COSMOZONE 2SA. The reconstruction showed that this cell had one irregularly
shaped nucleus and a wide ring-shaped clear zone and a small ruffled border. Under the ruffled border, this cell formed a small lacuna on the dentin
surface. The results suggested that this cell was a mononuclear odontoclast.
Conclusions: The present study concludes that cells with ruffled borders
and clear zones observed by transmission electron microscopy can be identified as odontoclasts or osteoclasts irrespective of the number of nuclei.
0 1994 Wiley-Liss, Inc.
Key words: Osteoclast, Ruffled border, Mononuclear cell, Multinucleation, Electron microscopy, Three-dimensional reconstruction
Osteoclasts have been considered multinucleated giant cells which possess special structures called ruff led
borders and clear zones observed by transmission electron microscopy (Scott and Pease, 1956; Schenk et al.,
1967; King and Holtrop, 1975; Wezeman et al., 1979;
Marks and Popoff, 1988). However, many investigators
have reported the presence of mononuclear osteoclasts
by light microscopy (Garant, 1976; Ries and Gong,
1982; Kaye, 1984; Marshall et al., 1986; Sire et al.,
1990; Athanasou et al., 1991; Prallet et al., 19921, by
transmission electron microscopy (Baron et al., 1986;
Sire et al., 1990), and by scanning electron microscopy
(Prallet et al., 1992). Some investigators have also
speculated on its theoretical presence as a precursor of
osteoclast (Chambers, 1985; Minkin and Shapiro, 1986;
Hattersley and Chambers, 1989).
0
1994 WILEY-LISS, INC.
With light microscopy, the presence of a ruffled border cannot be demonstrated conclusively. With transmission electron microscopy, a mononuclear cell apparently observed in one section may be multinucleated,
and with scanning electron microscopy, the presence of
both a ruffled border and single nucleus cannot be precisely demonstrated. Domon and Wakita (1991b) made
serial sections of cultured osteoclasts by alternating
semithin and ultrathin sections throughout whole cell
Received November 19, 1993; accepted March 21, 1994.
Address reprint requests to Dr. Takanori Domon, Department of
Oral Anatomy 11, Hokkaido University School of Dentistry, Kita 13,
Nishi 7, Kita-Ku, Sapporo 060 Japan.
MONONUCLEAR ODONTOCLAST WITH RUFFLED BORDER
bodies, and observed their ultrastructure by transmission electron microscopy and reconstructed the same
cells three-dimensionally from serial sections. As a result, Domon and Wakita (1991b) demonstrated the
presence of a mononuclear osteoclast with a ruffled
border in vitro. However, this mononuclear osteoclast
was merely a cultured cell originating from the mouse,
and the presence of mononuclear osteoclasts in vivo has
not been shown.
There are a number of difficulties in demonstrating
the presence of mononuclear osteoclasts in humans.
First, it is difficult to obtain intact human bone for
examination, and it may be diseased if it can be
obtained. Second, bone has many trabeculae with
complicated surface contours and it may be impossible
to find mononuclear osteoclasts or to make serial
sections throughout a complete cell body. To overcome
these drawbacks, the present study used human
deciduous teeth instead of bone. It is well known that
deciduous teeth are resorbed by osteoclast-like cells,
odontoclasts (Boyde and Lester, 1967; Furseth, 1968),
and that odontoclasts have the same structures as
osteoclasts (Furseth, 1968; Yaeger and Kraucunas,
1969; Freilich, 1971; Ohno, 1972; Suzuki, 1974;
Nilsen, 1977; Ten Cate and Anderson, 1986; Pierce et
al., 1991; Yawaka, 1993). Moreover, deciduous teeth
are easier to obtain than human bone because
deciduous teeth are naturally shed when the permanent teeth erupt.
To identify the odontoclasts on deciduous teeth by
light microscopy, the present study used histochemically TRACPase activity, which was considered specific
for both odontoclasts (Hasselgren and Stromberg, 1976;
Nilsen and Mugnusson, 1979; Yawaka, 1993) and osteoclasts (Hammarstrom et al., 1971; Minkin, 1982).
The present study aimed to demonstrate the presence
of a mononuclear odontoclast with a ruffled border.
43
room temperature. They were block-stained with 4%
uranyl acetate for 30 min, dehydrated in a graded series of ethanol, and then embedded in Epon 812. Specimens were sectioned in the direction perpendicular to
the resorbing surface and the cells for investigation
were serially sectioned with glass and diamond knives
on a Sorval MT-2B ultramicrotome by alternating
semithin and ultrathin sections throughout the width
of the cells. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (HITACHI H-7000) a t an
operating voltage of 75 kV.
Three-Dimensional Reconstruction
The semithin sections were stained with methylene
blue and azure 11, and then photographed by light
microscopy. The photographs were enlarged on the
printing paper, and the cell surface, dentin surface,
nucleus, ruffled border, and clear zone were traced
on tracing paper. Based on the outline of the section,
these elements were input serially into a personal
computer (NEC PC-9801 VM) with the Three-Dimensional Graphic Analytic System, COSMOZONE
2SA (NIKON, Japan). The three-dimensional reconstruction was calculated by computer, and the reconstructed image was photographed from display of
computer.
RESULTS
Histochemistry for TRACPase Activity
With a whole-mount light microscope, numerous
TRACPase-positive cells with different sizes and
shapes appeared dark on the resorbing surfaces in most
of the teeth (Figs. 1, 2). We observed TRACPase activity in all specimens and particularly looked for the
smallest TRACPase-positive cells, because mononuclear osteoclasts display small contours in vitro
(Domon and Wakita, 1991b). After observing the conMATERIALS AND METHOD
tours
of many TRACPase-positive cells in all speciHistochemistry for TRACPase Activity
mens, we selected a small area opposite to the crown
Twenty deciduous teeth from 6-10-year-old males (Fig. 2) of one lower right deciduous canine of an
and females were used in this study. The teeth were 8-year-old male for further investigation (Fig. 1).
extracted under local anesthesia a t Hokkaido UniverTo understand both the ultrastructure and three-disity Dental Hospital (Sapporo, Japan). After extrac- mensional features of TRACPase-positive cells, seven
tion, specimens were immediately fixed with 2.5% glu- TRACPase-positive cells of various sizes were selected
taraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) in this area (arrows 3-6 in Fig. 2).
overnight at 4"C, and then decalcified with 5% EDTA
(pH 7.4) for 2 months at 4°C. For the detection of
Light Microscopy
TRACPase activity, the azo dye method was used (BurThe
section
a
t
the
site of arrow 3 in Figure 2 showed
ston, 1958). Specimens were incubated in 0.1M acetate- two cells on the dentin
(Fig. 3). The left cell in Figure
buffered medium (pH 5.2) containing naphthol AS-MX 3 corresponded to the small
TRACPase-positive cell di(SIGMA, St. Louis, MO) as a substrate, and fast red rectly ahead of the point of arrow
3 in Figure 2, and the
violet (SIGMA) as a diazonium chloride, and 10 mM right cell in Figure 3 corresponded
the larger cell
sodium tartrate (SIGMA) (Minkin, 1982). Incubation immediately beyond it, to the right intoFigure
2. These
was carried out at 37°C for 10 min. Specimens for controls were incubated in the medium without a substrate under the same conditions. After the detection of
TRACPase activity, specimens were photographed
with a light microscope to determine the locations of
Abbreviations
TRACPase-positive cells on the resorbing surface of the cz
clear zone
D
dentin
teeth.
Light and Transmission Electron Microscopy
Specimens were postfixed with 1%osmium tetroxide
in 0.05M sodium cacodylate buffer (pH 7.4) for 3 hr at
ER
Mt
Nu
RB
V
rough-surfacedendoplasmic reticulum
mitochondria
nucleus
ruffled border
vacuole
44
T. DOMON ET AL.
Fig. 1. Whole mount preparation showing a lower right canine after
the detection of TRACPase activity. The lingual (arrowheads) and
mesial (M) root surface are resorbed extensively, and there are numerous TRACPase-positive cells on the dentin surface. Enamel was
decalcified, and only the dentin of the crown (C) is observed. A small
area (brackets) was selected for the observation of a mononuclear
odontoclast. Periodontal ligaments (P) are seen on the tooth. x 5. Bar
= 2mm.
Fig. 2. High magnification showing the bracketed part of Figure 1.
There are numerous TRACPase-positive cells on the dentin. The numbered arrows indicate the sites of the observations in Figures 3-6, and
two TRACPase-positive cells are indicated at each of the numbered
arrows 3, 5, and 6. x 80. Bar = 125 pm.
two cells had one nucleus in the cytoplasm and formed
lacunae on the dentin surface.
Figure 4 showed the TRACPase-positive cell indicated by arrow 4 in Figure 2. The cell had two nuclei in
its cytoplasm and fit in the lacuna in this section.
The section at the site of arrow 5 in Figure 2 showed
two cells on the dentin surface (Fig. 5). The left cell in
Figure 5 corresponded to the small TRACPase-positive
cell beyond the large TRACPase-positive cell a t the
point of arrow 5 in Figure 2. In this section, this cell
had one nucleus and many granules in its cytoplasm,
and formed a lacuna on the dentin. The right cell in
Figure 5 corresponded to the larger cell a t arrow 5.
This cell appeared larger and had several nuclei in the
cytoplasm.
Figure 6 showed two cells corresponding to the two
large TRACPase-positive cells to the right of the point
of arrow 6 in Figure 2. These two cells were multinucleated giant cells and formed lacunae on the dentin.
Observations of the serial sections of these cells allowed us to count precisely the number of nuclei in
them. The left cell in Figure 3 had one nucleus (Figs.
7-14), and the right cell had two; the cell in Figure 4
had two nuclei; the left cell in Figure 5 had three nuclei, and the right cell had eight; the left cell in Figure
6 had seven nuclei, and the right cell had five.
dentin surface (Fig. 15). The other multinucleated
TRACPase-positive cells showed similar ultrastructure. The right cell with two nuclei from the observations of the serial sections in Figure 3 also had a typical
ruffled border and clear zone facing the dentin surface.
Figures 16-19 showed the serial ultrathin sections of
the small TRACPase-positive cell in Figures 3 and
7-14. Figure 16 showed the section 2 pm from the appearance of the cell body. There were two cytoplasmic
parts with many microvilli in this section.
In the section 4 pm from the cell surface (Fig. 17),
the cellular profiles were larger and there were
many mitochondria, rough-surfaced endoplasmic reticulum, and vacuoles in the cytoplasm. The clear zone
was seen on the dentin surface, but there was no
ruff led border.
Seven micrometers from the cell surface (Fig. 181,
the cellular profiles were again larger. There was one
nucleus in the central area of the cytoplasm and developed Golgi apparatus surrounding it, and a large number of mitochondria, rough-surfaced endoplasmic reticulum, and vacuoles were seen in it. This cell had a
ruffled border and clear zone (Figs. 18, 201, and the
cytoplasmic processes composing the ruff led border
were both finger- and plate-like. On the dentin surface
under the ruffled border there was a small lacuna
where the collagen fibers of the dentin were disrupted
(Fig. 20).
Fen micrometers from the cell surface (Fig. 191, the
cellular profiles were smaller and no nucleus was observed in the cytoplasm. Only the clear zone was seen
on the dentin, and no ruffled border was observed.
There was no dentin disruption under the clear zone.
Transmission Electron Microscopy
The left cell in Figure 6 showed several nuclei with
the Golgi apparatus surrounding these, as well as numerous mitochondria, rough-surfaced endoplasmic
reticulum, and vacuoles in the cytoplasm (Fig. 15).
This cell had a ruffled border and clear zone facing the
MONONUCLEAR ODONTOCLAST WITH RUFFLED BORDER
Fig. 3-6. Light micrographs showing semithin sections of TRACPase-positive cells sectioned a t arrows 3-6 in Figure 2. Figs. 3 and 4,
x 1,100.Figs. 5 and 6, x 1,000,Bar = 10 pm.
Fig. 3. Two cells have revealed resorptive lacunae (arrowheads) on
the dentin.
45
Fig. 5. A cell with numerous granules and multinucleated cell are
related to resorptive lacunae (arrowheads) on the dentin.
Fig. 6. Two multinucleated cells are associated with resorptive lacunae (arrowheads) on the dentin.
Fig. 4. Rounded cell occupies the resorptive lacuna (arrowheads).
Three-DimensionalReconstruction
The small TRACPase-positive cell in Figure 3 was
reconstructed from the serial sections in Figures 7-14.
These reconstructions included the dentin interface
with the cellular surface, nucleus, ruffled border, and
clear zone. When the cellular and dentin surfaces were
displayed together (Fig. 21), the cell showed a rounded
gourd-shaped outline with a total width of about fourteen micrometers. When the nucleus was added to the
reconstruction (Fig. 22), one irregularly shaped nucleus was observed in the central area of the cytoplasm,
and there was a small lacuna on the dentin surface
under the cell. The diameter of the lacuna was about 6
Fm. When the clear zone and ruff led border were added
(Fig. 23), the clear zone formed a wide ring-shaped
structure encircling a small area of ruffled border. The
area of the ruffled border was relative to that of lacuna
in Figure 22. All elements were displayed together in
Figure 24; this cell was shown to possess the one nucleus and a small ruffled border surrounded by a wide
clear zone on the dentin surface. This cell overlay a
small lacuna.
DISCUSSION
The present study used the azo dye method to visualize the light microscopic TRACPase-positive cells on
the resorbing surface of human deciduous teeth. Most
of the TRACPase-positive cells were multinucleated
with more than two nuclei, possessing ruffled borders
and clear zones. These cells formed lacunae on the dentin surface under the ruffled border. These characteristic features are the same as those of odontoclasts observed by transmission electron microscopy (Furseth,
1968; Yaeger and Kraucunas, 1969; Freilich, 1971;
Ohno, 1972; Suzuki, 1974; Nilsen, 1977; Ten Cate and
Anderson, 1986; Pierce et al., 1991, Yawaka, 1993),
and it is concluded that TRACPase-positive cells on the
deciduous teeth are odontoclasts.
The precursor of osteoclast or odontoclast has been
thought to be the preosteoclast or preodontoclast, re-
46
T. DOMON ET AL.
figs.7-14. Serial semithin sections of the left cell in Figure 3 taken, respectively, at 1 , 3 , 4 , 5 , 6 , 8 , 9 ,
11, and 12 pm from the cell surface. The serial sections show that the cell has only one nucleus (arrows)
in the cytoplasm and that it forms a resorptive lacuna (asterisks) on the dentin. A leukocyte (L) is
observed near this cell (Fig. 7). x 1,500. Bar = 7 pm.
The mononuclear odontoclast in the present study
spectively. It has been reported that the preosteoclasts
are mononuclear cells with some of the characteristic showed a rounded outline in three-dimensions. It has
features of osteoclasts; however, they do not possess been shown that actively resorbing osteoclasts have
any ruffled border conclusively (Scott, 1967; Luk et al., rounded contours and that migrating ones have flat
1974; Rifkin et al., 1980; Ejiri, 1983). Preodontoclasts and irregular shapes in vitro (Domon and Wakita,
with TRACPase activity were not observed on the de- 1991a). Although the reconstructed cell was present in
ciduous tooth in this study. However, we have observed vivo, the results would allow the mononuclear odontoTRACPase-positive cells originating from long bone of clast in the present study to be classified as a resorbthe mouse on dentine slices in vitro, and reported that ing-type odontoclast. In fact, the three-dimensional reTRACPase activity was detected in both osteoclasts construction showed that this cell had a ring-shaped
and preosteoclasts (Domon and Wakita, 1991b). Con- clear zone encircling a small ruffled border, and formed
sidering the fact that preosteoclasts also possessed a small lacuna.
It has been reported that 80% of human odontoclasts
TRACPase activity in vitro, it is possible that mononuclear preodontoclasts may be present among many (Addison, 1978) and 81% of chick osteoclasts (Piper et
TRACPase-positive cells on the resorbing areas of al., 1992) have ten or fewer nuclei. In the present
study, we also counted the number of nuclei in odontoteeth.
The present study showed that one mononuclear clasts and found that all of them had eight or fewer
TRACPase-positive cell possessed a typical ruff led bor- nuclei. Addison (1978) and Piper et al. (1992) deliberder and clear zone by transmission electron micros- ately excluded the mononuclear cells from their analcopy. Its ultrastructural features were similar to those yses of the distribution of nuclei because there had
of multinucleated odontoclasts, and the three-dimen- been no conclusive evidence of the presence of monosional reconstruction showed that this cell had only one nuclear odontoclasts and osteoclasts. The present and
irregularly shaped nucleus. Therefore, the ultrastruc- our recent study (Domon and Wakita, 1991b) have
ture and three-dimensional structure suggest that this shown the presence of mononuclear odontoclasts and
TRACPase-positive cell is a mononuclear odontoclast. osteoclasts with the ruffled borders. Considering these
MONONUCLEAR ODONTOCLASTWITH RUFFLED BORDER
47
Fig. 15. Eleclrun micrograph of the left odontoclast in Figure 6 . The multinucleated odontoclast has a
ruffled border (inset)and clear zone on the dentin. There is a Golgi apparatus (Go),many mitochondria,
rough-surfaced endoplasmic reticulum, and vacuoles in the cytoplasm. ~3,600,Bar = 3 pm. Inset:
x 11,000, Bar = 1 pm.
facts, we suggest that most osteoclasts and odontoclasts
are the cells with a small number of nuclei, and that
these small osteoclasts and odontoclasts play an important role in hard tissue resorption.
Hattersley and Chambers (1989) speculated that the
osteoclast was initially mononuclear and might remain
so, but it generally became multinucleated. The
present study showed the presence of mononuclear odontoclasts. This fact suggests that the odontoclast may
be initially mononuclear. We were not able to establish
whether the mononuclear odontoclast became multinucleated or not. Mononuclear odontoclasts may have two
roles, the resorption of the tooth and the recruitment of
precursors for multinucleation. The origin of mononuclear odontoclasts also remains unknown.
Kolliker (1873) observed multinucleated giant cells
in Howship’s lacunae on bones by light microscopy, and
called these multinucleated cells “Ostoklasten,” or osteoclasts. After this report, multinucleated cells seen
on bone by light microscopy have generally been
thought to be osteoclasts. Scott and Pease (1956) were
the first to observe osteoclasts by transmission electron
microscopy. They reported that bone was resorbed by
special structures of osteoclasts, and called these the
ruffled borders. Therefore, the cells with ruffled borders observed by transmission electron microscopy
have been considered to be osteoclasts.
Besides multinucleation and the presence of a ruffled border, many characteristics of osteoclasts have
been proposed: the presence of a ruffled border and
clear zone (Dudley and Spiro, 1961; Schenk et al., 1967;
Kallio et al., 1971; King and Holtrop, 1975; Wezeman
et al., 1979; Marks and Popoff, 1988), TRACPase activity (Hammarstrom et al., 1971; Minkin, 1982; Baron et
al., 1984; Andersson et al., 1986; Glowacki and Cox,
1986; Helfrich et al., 1989; Takahashi et al., 1988; Udagawa et al., 19901, calcitonin receptors (Warshawsky
et al., 1980; Chambers and Magnus, 1982; Arnett and
Dempster, 1987; Hattersley and Chambers, 1989), carbonic anhydrase (Gay and Mueller, 1974; Anderson et
al., 19821, ATPase (Baron et al., 19851, vitronectin receptors (Davies et al., 19891, and cellular surface antigens different from those of macrophage-monocyte lineage cells (Horton et al., 1985; Nijweide et al., 1985;
Athanasou et al., 1991; Collin-Osdoby et al., 1991).
The nomenclature of osteoclast and odontoclast applies generally, based on the hard tissue substrate
which is resorbed; osteoclasts resorb bone and odontoclasts resorb tooth. Previously, the multinucleated giant cells resorbing deciduous teeth had been also called
osteoclasts by light microscopy (Kronfeld, 19321, and
Furseth (1968) was the first to observe them by transmission electron microscopy, and has referred to them
as odontoclasts. Accordingly, it has been also known
that odontoclasts are multinucleated giant cells which
have the same characteristic features as osteoclasts:
the presence of the ruffled border and clear zone (FurSeth, 1968; Yaeger and Kraucunas, 1969: Freilich,
1971; Ohno, 1972; Suzuki, 1974; Nilsen, 1977; Ten
Cate and Anderson, 1986; Pierce et al., 1991; Yawaka,
Figs. 16-1 9. Electron micrographs showing serial ultrathin sections
of the mononuclear odontoclast in Figures 3 taken, respectively, at 2,
4, 7, and 10 pm from the cell surface. X 3,000. Bar = 4 pm.
Fig. 16. Two areas of the cytoplasm are observed in this site. There
is a leukocyte (L) near the dentin.
Fig. 17. Cytoplasmic areas are larger, showing many mitochondria,
rough-surfaced endoplasmic reticulum, and vacuoles. Only the clear
zone is seen next to the dentin.
Fig. 18. The cell has one nucleus, and the ruffled border and clear
zone are seen at the dentin surface. There is a small lacuna (asterisk)
under the ruffled border. Most of the rough-surfaced endoplasmic reticulum occurs between the nucleus and the Golgi apparatus (Go).
Many mitochondria and vacuoles are observed in the cytoplasm.
Fig. 19. There is no nucleus present in this section, and only the
clear zone is seen next to the dentin. Many mitochondria, roughsurfaced endoplasmic reticulum, and vacuoles are present in the cytoplasm.
MONONUCLEARODONTOCLASTWITHRUFFLEDBORDER
49
Fig. 20. Electron micrograph showing a part of Figure 18 a t a higher magnification. The ruffled border
surrounded by the clear zone is seen against the dentin surface. Under the ruffled border, there is a small
lacuna (asterisk), and the collagen fibers composing the dentin are disrupted. x 12,000. Bar = 1 pm.
1993) and TRACP activity (Hasselgren and Stromberg,
1976; Nilsen and Mugnusson, 1979; Yawaka, 1993).
Therefore, osteoclasts and odontoclasts would be called
"elastic" cells for hard tissue resorption. In fact, osteoclasts can also resorb enamel and dentin which are
components of tooth in vitro (Jones et al., 1984).
Although many characteristics of osteoclasts and odontoclasts have been proposed, the conclusive criterion
for identification of a cell as an osteoclast or odontoclast has been considered the presence of a ruffled
border and clear zone observed by transmission electron microscopy (Scott and Pease, 1956; Schenk et al.,
1967). With this criterion, many investigators have
identified cells with ruffled borders and clear zones as
osteoclasts or odontoclasts, and in the present study we
identified the mononuclear cell with these features as a
mononuclear odontoclast. However, osteoclasts and
odontoclasts have been generally considered to be
multinucleated giant cells, and not mononuclear cells
(Kolliker, 1873).
The significance of the multinucleation of osteoclasts
and odontoclasts must be better understood before it is
possible to identify the mononuclear cell with a ruff led
border as an odontoclast. Based on light microscopic
observations, multinucleation has been proposed as
one criterion for osteoclast identification (Kolliker,
1873). The presence of a ruffled border, the other criterion, has been based on transmission electron microscopic observation (Scott and Pease, 1956). Therefore,
considering the conditions of observation, these two criteria are quite different. If osteoclasts and odontoclasts
must always satisfy both criteria, the mononuclear cell
with a ruff led border in this study cannot be classified
as either.
Hattersley and Chambers (1989) suggested that
multinuclearity was an unreliable marker for the
osteoclastic phenotype in culture. Prallet et al. (1992)
also speculated that multinucleation might not be
necessary for full expression of the osteoclast function
in bone resorption. Moreover, multinucleated cells
observed by light microscopy are not always osteoclasts or odontoclasts. It is known that multinucleated
giant cells encircle bone particles in vivo (Popoff and
Marks, 1986). Macrophages have similar structures to
those of osteoclasts, and the clear zones are seen in
both macrophages and osteoclasts; however, macrophages lack a ruffled border at the interface between
the cell and the hard tissue substrate (Kahn et al.,
1978; Rifkin et al., 1979; Popoff and Marks, 1986).
Therefore, the conclusive criterion to identify a cell as
an osteoclast or odontoclast is the presence of a ruffled
border and clear zone observed by transmission
electron microscopy.
Our recent study (Domon and Wakita, 1991b) and
the present study have demonstrated the presence of a
mononuclear cell with a ruffled border and clear zone
both in vitro and in vivo. Therefore, we conclude that
cells with ruffled borders and clear zones observed by
transmission electron microscopy can be identified as
osteoclasts or odontoclasts, irrespective of the number
of nuclei. However, this conclusion does not apply to
mononuclear cells observed by light microscopy, because a t this level it is not possible to conclusively demonstrate the presence of a ruffled border. A further
50
T. DOMON ET AL.
Figs. 21-24. Models showing the three-dimensional structure of the
mononuclear odontoclast. These images are displayed as looking obliquely to the resorbing surface. The odontoclast (Ocl) shows a
rounded gourd-shaped outline on the dentin (Fig. 21). When the cell
surface is displayed as semitransparent (Figs.22-24), one irregularly
shaped nucleus is seen in the central area of the cytoplasm, and a
lacuna (La) can be seen on the dentin under the cell (Fig. 22). A wide
ring-shaped clear zone and small ruffled border are present on the
dentin (Fig. 23). Displaying all structures together allows a n understanding of the three-dimensional arrangement (Fig. 24). x 2,000.
Bar = 5 pm.
evaluation of multinucleation as a criterion of both osteoclasts and odontoclasts is needed.
Arnett, T.R., and D.W. Dempster 1987 A comparative study of disaggregated chick and rat osteoclasts in vitro; effects of calcitonin
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Athanasou, N.A., B. Puddle, J . Quinn, and C.G. Woods 1991 Use of
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Baron, R., L. Neff, D. Louvard, and P.J. Courtoy 1985 Cell-mediated
extracellular acidification and bone resorption: evidence for a low
pH in resorbing lacunae and localization of a 100-kD lysosomal
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175-234.
Baron, R., P. Tran Van, J.R. Nefussi, and A. Vignery 1986 Kinetic and
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368-378.
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ACKNOWLEDGMENTS
We thank Y. Honma, Department of Oral Anatomy
11, Hokkaido University School of Dentistry, for his
technical assistance; and C.M.T. van de Sande-Rijkers,
Laboratory of Cell Biology and Histology, University of
Leiden, The Netherlands, for stimulating suggestions
on this study. This study was supported by Grant-inAids for Scientific Research from the Japanese Ministry of Education, Science and Culture (03771263 and
04454449).
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